The ZDSD Rat as a Translational Model for the Development of Drugs for Obesity, Metabolic Syndrome and Diabetes. This presentation describes the ZDSD rat. This rat is a model of obesity, metabolic syndrome and diabetes. In the pre-diabetic state it demonstrates all of the characteristics of human metabolic syndrome including obesity, glucose intolerance, insulin resistance, increased cardiovascular biomarkers and hypertension. In the diabetic state it expresses diabetic complications such as diabetic nephropathy, osteoporosis and delayed wound healing.
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INDEX 1. Introduction Page 2- 2. Spontaneous or Synchronous Diabetes Page 7- 3. Metabolic Syndrome Elements Page 10- A. Visceral Obesity Page 13- B. Insulin Resistance Page 19- C. Clamp Study Page 28- D. Dyslipidemia Page 32- E. Hypertension Page 35- 4. Eating Behavior Page 37- 5. Beta cell Failure Page 38- 6. Renal Injury Page 41- A. Urinary biomarkers, Exp. 1, 2 Page 43- B. RBM Biomarkers – Renal, Exp 3 Page 54- C. Glomerular Pathology, EM, Exp 4 Page 63- D. Synchronized Nephropathy, Exp 5 Page 72- 7. Osteoporosis Page 81- 8. Wound Healing Page 87- 9. RBM Biomarkers – Pro-Thrombotic Page 90- 10. RBM Biomarkers - Inflammation Page 96- 11. Therapeutic Efficacy A. Common Anti-diabetic Compounds Page 104- B. Rimonabant Page 109- C. Niacin Page 117- 12. Summary Page 115- 1 Slide →
Transcript
INDEX1. Introduction Page 2-2. Spontaneous or Synchronous Diabetes Page 7-3. Metabolic Syndrome Elements Page 10-
A. Visceral Obesity Page 13-B. Insulin Resistance Page 19-C. Clamp Study Page 28-D. Dyslipidemia Page 32-E. Hypertension Page 35-
A. Common Anti-diabetic Compounds Page 104-B. Rimonabant Page 109-C. Niacin Page 117-
12. Summary Page 115- 1Slide →
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The ZDSD Rat as a Translational Model for the Development of Drugs for Obesity, Metabolic
Syndrome and Diabetes that Demonstrates Many of the Serious Complications of Diabetes.
PreClinOmics, Inc.
2
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• Most rodent models of type 2 diabetes have a monogenetic mutation that is responsible for the initiation of obesity and subsequent insulin resistance.
• The two most common obesity-causing mutations are– the leptin receptor
• Zucker Fatty; ZF rat
• Zucker Diabetic Fatty; ZDF rat
• db/db mouse
– the leptin molecule
• ob/ob mouse
• Both leptin and leptin receptor mutations are rare in humans.
• The ZDSD rat does not have these mutations but still has obesity metabolic syndrome and diabetes.
Background
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Development SchemeZucker Diabetic-Sprague Dawley Rat (ZDSD)
• Produced by crossing diet induced obese (DIO) rat derived from the Crl:CD(SD) strain (exhibiting polygenetic obesity and insulin resistance) with homozygous lean ZDF/Crl rat (which will express beta cell failure with the Leprfa/Leprfa genotype).
• Selectively bred for obesity and diabetes.
• Selected for genetically matched breeders to develop phenotypic homogeneity.
• Studied male rats at different ages.
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Characteristics of The ZDSD Rat• Unique translational model for obesity, metabolic
syndrome/type II diabetes - 35 generations inbred
• Polygenic obesity and phenotype can be modulated by diet.
• Phenotype is expressed in the presence of a functional leptinpathway.
• Insulin resistance development starts at an early age.
• Early onset of hyperglycemia and slower progression to frank diabetes when compared to the ZDF rat.– Slower deterioration of beta cell function.
maintained on Lab Diet 5008 chow will spontaneously develop diabetes as it ages beyond 16 wks. As fed serum glucose levels begin to increase above ~350 mg/dl, body weight begins to decrease.
Synchronization of diabetic onsetThe ZDSD Rat can be placed
on either D12468 (Research Diets) or 5SCA (LabDiet) to synchronize the onset of diabetes.
When the ZDSD rat was
placed on either diet at 17 wks of age, the plasma glucose levels of the animals averaged over 450 mg/dl within 1 week. Following a return to LabDiet 5008 at 19 wks of age, the animals maintained the diabetic state.
PCO now recommends a 3 week synchronization protocol
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
0
200
400
600
SD Male Rats
ZDSD Males
ZDSD Females
Age (wks)
Glu
co
se (
mg
/dl)
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
0
200
400
600
SD Male Rats
ZDSD Males
ZDSD Females
Age (wks)
Weig
ht
(g)
Area shaded in grey indicates time frame of diabetogenic diet
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Body composition changes in response to diabetogenic diet (5SCA or D12468).
% Body Fat by QNMR
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
0
5
10
15
20
25
30
SD Male Rats
ZDSD Males
ZDSD Females
Age (wks)
% B
od
y F
at
Area shaded in grey indicates time frame of diabetogenic diet
Synchronization of diabetic onset
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Metabolic syndrome affects a large proportion of the population and is becoming
increasingly important in adolescents. The syndrome has many components
including central obesity, insulin resistance, dyslipidemia and hypertension. In
addition, the syndrome features a chronic low grade inflammatory state, vascular
endothelial dysfunction, and a prothrombotic environment. Long standing
metabolic syndrome can thus pre-dispose to atherosclerosis, microvasculature
disease (retina), stroke, renal injury and diabetes. Due to the complicated
mechanisms involved in the syndrome and its sequelae, current standard of care
reflects poly-pharmacy and is aimed at controlling atherogenic dyslipidemia,
hyperglycemia and hypertension as well as intervening in secondary diseases such
as renal dysfunction, stroke, and micro-vascular disease related to retinopathy.
Development of new chemical entities with the potential to control more than one
risk factor is hampered by currently available animal models. To that end, the ZDSD
rat was designed to spontaneously develop a phenotype that mimics many aspects
of the human metabolic syndrome, including hypertension and the progression to
frank diabetes with long-standing disease.
ZDSD as a preclinical model of Metabolic Syndrome
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Metabolic Syndrome
• Metabolic syndrome is most frequently defined by a presence of certain traits, including:
– abdominal obesity
– insulin resistance
– Dyslipidemia
– elevated blood pressure and
– pro-thrombotic and pro-inflammatory states
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Characteristics of Metabolic Syndrome Seen in the ZDSD Rat
• Increased body weight with increased abdominal fat
• Insulin resistance / Glucose intolerance
• Hyper-lipidemia
• Increased blood pressure / Hypertension
• Increased Serum BioMarkers of Coagulation inflamation and Vascular Disease
• Increased fed and fasting glucose and HbA1c levels Return to Index
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A prominent component of metabolic syndrome is insulin resistance which is thought to be mediated by an increase in metabolically active visceral fat. Visceral fat accumulation occurs in human patients in the presence of a functional leptin
pathway as leptin deficiencies and receptor defects are rarely reported. According to published growth charts for male leptin resistant ZDF rats, the new ZDSD rats are
heavier when fed a normal diet (PMI 5008) and exhibit a body composition (increased % fat) comparable to age matched DIO-LE model which is a mainstay for anti-obesity research. In addition, the ZDSD responds to a common reference anti-obesity agent (rimonabant) with significant loss of body fat. Interestingly, ZDSD rats
are not typically nocturnal in that they exhibit significant feed intake during the daylight hours. Exogenously administered leptin results in an acute anorexic effect quite similar to normal SD rats and indicates the presence of a functioning leptin
pathway
Visceral Obesity
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Comparative Growth Curvesin SD and ZDSD Rat Fed 5008 chow
ZDSD rats were significantly (15%) heavier than their SD counterparts at 8 weeks of age. In addition, the rate of body weight gain was increased in ZDSD animals as evidenced by an 82% vs 62% weight gain in SD animals during the 24 weeks.
Study # 09-550-170
All time points statistically different
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Spontaneous Development of Obesity in ZDSD Rats Fed 5008 Chow
Study # 09-550-170
All time points statistically different
Body composition was assessed using QNMR . The percentage of body weight identified as fat was 50 % higher in ZDSD compared to SD controls as early as 8 weeks of age. Body fat percentage continued to increase throughout the study and remained significantly higher than control rats at each time-point.
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Visceral Obesity in the ZDSD RatCT Scan
Sub-cutaneous fat
Retroperitoneal fat
Visceral fat
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Terminal Body Weight Comparison
Terminal animal weights in diabetic and control animals.
100
200
300
400
500
600
700CRL-SD, CD
+/fa
ZDF
ZDSD, Diabetic 12-21 weeks
ZDSD, Diabetic 7-11 weeks
Weig
ht (g
)
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Terminal ComparisonLiver Weight Food & Water Consumption
Terminal liver weights, water intake and food consumption are highest in the diabetic groups.
Development of Insulin Resistance in the ZDSD Rat on 5008 Purina chow
• Rats tested started at 8 weeks of age (SD & ZDSD)
• Weight, glucose and insulin measured weekly
• Animals fasted every two weeks for OGTT
• Data analyzed– Weight
– Body composition
– Glucose levels
– OGTT glucose and insulin
– Glucose disposal
– HOMA-IR
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Comparative Growth Curvesin SD and ZDSD Rat Fed 5008 chow
ZDSD rats were significantly (15%) heavier than their SD counterparts at 8 weeks of age. In addition, the rate of body weight gain was increased in ZDSD animals as evidenced by an 82% vs 62% weight gain in SD animals during the 24 weeks.
Study # 09-550-170
All time points statistically different
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Spontaneous Development of Obesity in ZDSD Rats Fed 5008 Chow
Study # 09-550-170
All time points statistically different
Body composition was assessed using QNMR . The percentage of body weight identified as fat was 50 % higher in ZDSD compared to SD controls as early as 8 weeks of age. Body fat percentage continued to increase throughout the study and remained significantly higher than control rats at each time-point.
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Spontaneous Development of Hyperglycemia in ZDSD Rats Fed 5008 Chow
Study # 09-550-170
All time points statistically different
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Spontaneous Development of Glucose Intolerance Shown by OGTT in ZDSD Rats Fed 5008 Chow
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Spontaneous Development of Insulin Resistance Shown by OGTT in ZDSD Rats Fed 5008 Chow
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Spontaneous Development of Impairment in Glucose Disposal in ZDSD Rats as Demonstrated by OGTT, AUC
Study # 09-550-170
Impairment in glucose disposal as represented by the area under the glucose curve during an oral glucose tolerance test developed spontaneously in ZDSD rats and was evident as early as 8 weeks of age (fed Purina 5008 chow).
All time points statistically different
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ZDSD rats become increasingly more insulin resistant with age as evidenced by the calculated HOMA-IR. The insulin resistance is evident compared to SD rats as early as 8 weeks of age (fed Purina 5008 chow).
Progressive Development of Insulin Resistance (HOMA-IR) in ZDSD Rats
All time points statistically different
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Glucose in CD vs ZDSD16 weeks of age
CD
ZDSD
100
110
120
130
140
150CD
ZDSD
Blo
od
Glu
co
se m
g/d
L
Glycated Hb in CD vs ZDSD16 weeks of age
CD
ZDSD
3.0
3.2
3.4
3.6
3.8
4.0CD
ZDSD
Gly
cate
d H
b
Glucose and Glycated Hemoglobin Levels in CD and Prediabetic ZDSD Rats
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Pre-diabetic Insulin Sensitivity,
hyperinsulinemic-euglycemic glucose
clamp
Design
– Rosiglitazone treatment: 3 mg/kg PO, QD for 2 weeks
– Comparison of insulin sensitivity at 9 wks of age in• The ZDSD Rat,
• Zucker Fatty (ZF), and
• Sprague Dawley (SD) rats
– Assessed by exogenous glucose infusion rate (GIR) during hyperinsulinemic (25 mU/kg/min)-euglycemic glucose clamp
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Body Weight and Basal Glucose of Rats Before Undergoing Glucose Clamp
* P<0.05 compared to SD rat (age matched) group# P<0.05 compared to vehicle treated group
SD (age) SD (wt) ZDSD ZF0
100
200
300
400
500
#
Rat Strain (SD rats are age or weight matched)
Bo
dy W
eig
ht
(g)
SD (age) SD (wt) ZDSD ZF0
40
80
120
160
200Vehicle (n=6-8)
Rosiglitazone (3 mg/kg PO, n=6-8)
Rat Strain (SD rats are age or weight matched)
Ba
sa
l B
loo
d G
luco
se
(m
g/d
l)
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ZDSD and ZF Rats are Insulin Resistant which
Improved with Rosiglitazone Treatment
* P<0.05 compared to SD rat (age matched) group# P<0.05 compared to vehicle treated group
SD (age) SD (wt) ZDSD ZF0
10
20
30
40
50
60 Vehicle (n=6-8)
Rosiglitazone (3 mg/kg PO, n=6-8)
#
#
Rat Strain (SD rats are age or weight matched)
Glu
co
se
In
fusio
n R
ate
(mg
/kg
/min
)
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ZDSD Rats Have Modest Visceral and
Whole Body Obesity Unlike ZF Rats
* P<0.05 compared to SD rat (age matched) group# P<0.05 compared to vehicle treated group
SD (age) SD (wt) ZDSD ZF0
2
4
6
8
10
12Vehicle (n=6-8)
Rosiglitazone (3 mg/kg PO, n=6-8)
#
Rat Strain (SD rats are age or weight matched)
Ep
idid
ym
al F
at
Pa
d (
g)
SD (age) SD (wt) ZDSD ZF0
10
20
30
40
Vehicle (n=6-8)
#
Rat Strain (SD rats are age or weight matched)
Bod
y F
at
(%)
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Patients with Type II diabetes and metabolic syndrome often present with dyslipidemia including elevated cholesterol and triglycerides and decreased HDL-
C. These lipids have been shown to impact cardiovascular and renal co-morbidities. Hypertriglyceridemia expresses as early as 12 weeks of age in ZDSD
rats when maintained on a normal diet and levels progress up to 500 mg/dL by 15 weeks. Similar to the fructose fed rat, a model commonly used for the study of
dyslipidemia, the spontaneous nature of the ZDSD lipid abnormality may provide a relevant model for the examination of compounds affecting the up-regulated lipogenic pathway seen in metabolic syndrome. The dyslipidemia in this model
responds to classic reference agents including rosiglitazone. Increases in cholesterol are not as dramatic and may be induced by feeding a high fat diet
Dyslipidemia
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Terminal Comparison of Models
Glu
cose TG
CHOL
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
An
aly
tes (
mg
/dL
)
Glucose, triglyceride and cholesterol levels. Glucose, triglyceride and cholesterol levels are elevated in all of the diabetic groups (ZDF and ZDSD). The model and duration of diabetes did not have a
significant effect on these measurements.Return to Index
High blood pressure is a key symptom of metabolic syndrome and is a major contributor to the increased risk of cardiovascular disease, kidney disease and
ischemic stroke seen in these patients. Examination of the interactions of all the components of the syndrome in rats is complicated by the absence of high blood pressure in current models (i.e., Zucker fatty rat). Indirect evidence of probable
elevated pressure in the form of elevated biomarkers for an activated RAAS ,endothelial dysfunction and aberrant vasoconstriction is noted in ZDSD rats.
Direct evidence of Hypertension has been confirmed in the pre-diabetic state via the tail-cuff method.
Hypertension
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Blood pressure data produced in collaboration with Dr. Subah Packer’s Laboratory, IU School of Medicine
60 70 80 90 10080
100
120
140
160ZDSD
CD
Age in Days
Systo
lic B
P
Blood pressure in ZDSD vs CD Rats
8-16 weeks of age
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Leptin PhysiologyFood intake of ZDSD Rats is
more evenly divided between day and night
Food Intake of ZDSD Rats is
Reduced in Response to Leptin Indicating a Functioning Leptin Pathway. Leptin was given just before the start of the dark cycle and food intake was measured for the first 4 dark hours.
SD ZDSD
Fo
od
in
take
(g/p
erio
d)
0
5
10
15
20
25
30
dark cycle
light cycle
daily total
ANOVA/pooled t (p<0.05) compared
to SD animals
Assessment of leptin pathway function as determined by feeding response to leptin injection (1 mg/kg, IP)
SD-saline SD-leptin ZDSD-saline ZDSD-leptin
Fo
od
in
take
4 h
rs a
fte
r tr
ea
tme
nt
0
1
2
3
4
5
6
7
8
9
10ANOVA/pooled t (p<0.05) compared to corresponding saline control
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Insulin Levels Decline as Diabetes Progresses
Glucose Insulin
5 7 9 11 13 15 17 19 21 23 25 27 29
0
1
2
3
4
5
6
7
8
11-13 WEEKS
15 WEEKS
17 WEEKS
Age (wks)
Insu
lin
(n
g/m
l)
Insulin levels of the group that become diabetic between 11-17 weeks of age. The animals that become diabetic earlier have higher insulin levels than those who become diabetic later.
The glucose levels for ZDSD rats were followed from 16 to 28 weeks of age (upper figure). At 28 weeks of age approximately 75% of the animals were overtly diabetic. The average glucose levels for each animal (16 to 28 weeks) were correlated with insulin content of the pancreas when the animals were terminated at about 28 weeks of age (lower figure). Higher average glucose levels were associated with lower insulin content in the pancreas.
Pancreatic Insulin Content
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Representative Islets from ZDSD Rats
Pre-diabetic Diabetic
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Obesity and metabolic syndrome are clear predictors of chronic kidney disease largely due to the
potentiation of chronic inflammation by insulin resistance. In addition, the lipoprotein abnormalities,
increased hemodynamics, hypercoagulability and vascular dysfunction associated with metabolic
syndrome have all been implicated as causative for renal disease. Biomarkers for renal dysfunction (i.e.,
IL6, TNF-α,NGAL,KIM-1, VEGF etc.) as well as significant albuminuria , elevated free fatty acids with oxidative stress, and histological analysis have shown the ZDSD
rat to exhibit nephropathy that closely mimics that observed in obese insulin resistant patients.
Renal Injury
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Terminal kidney weights are highest in the ZDSD Rat groups. These increased kidney weights and high urinary volume along
with increased micro-albumin concentration and the total amount of micro-albumin indicate that there may be significant diabetic nephropathy in the ZDSD Rat model.
spontaineously on Purina 5008 and aged to 33 weeks. Two groups of animals were selected for further study: animals that were diabetic for longer than 16 weeks and animals that were diabetic for less than 8 weeks.
• Mesoscale (MSD) urine panels were run on urine (ArgutusAKI test, Kidney Injury Panel 1 and Rat Clusterin)
• Pathological evaluation of the kidneys was done.
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Data From Urinary Excretion Study
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Urinary Excretion of Kidney Markers
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Urinary Excretion of Kidney Markers
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Pathological Evaluation of Kidney• Glomerulopathy: Changes in the renal glomeruli consisted of one or more of the following: increased
cellularity in the mesangium; increased in mesangial connective tissue; thickening of Bowman’s capsule; hypertrophy of capsular epithelium; dilation of the capsular space. Individual glomeruli appeared moderately enlarged. The lesions were highly variable within individual glomeruli and between glomeruli within a kidney. The changes were most usually segmental, although a rare glomeruli was fibrotic (condensed). Expanded mesangial material stained positively with the PAS stain and to a lesser extent with the Trichrome stain.
• Tubular dilation/degeneration: This change was mainly in the cortex and consisted of irregularly dilated, empty tubules, that sometimes were lined by cuboidal epithelium that stained basophilic compared to the expected normal eosinophilic tubular epithelium. In some individual tubules the epithelium were flattened. These dilated/degenerate tubules were randomly scattered throughout the cortex, and sometimes were associated with protein casts and/or non-suppurative inflammation (see below). Focal mild increases in fibrous connective tissue within the interstitial space was present, frequently in association with the interstitial inflammatory response, but not restrictively so.
• Protein casts: Individual tubules contained acellular, uniformly staining eosinophilic material consistent with protein. These protein casts were present in the cortex and in the medulla, as well as at the cortico-medullary junction in various sections. Often, several such dilated tubules containing protein casts were clustered together, usually in the cortex.
• Inflammation: The inflammatory process consisted of focal collections of lymphocytes and macrophages, which were seen in the cortical interstitial space, adjacent to individual glomeruli and individual blood vessels, and in association with the renal pelvic epithelium.
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Glomerulopathy Tubular dilation Protein casts Inflammation
His
top
ath
olo
gy S
co
re (
0-5
)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0non-diabetic
diabetic compared to Non-diabetic animals (t-test)
/degeneration
Kidney Histopathology of the ZDSD Rat
A Novel Animal Model of Diabetes
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Experiment 3Serum BioMarkers of Renal Disease
RBM Collaboration
Rules Based Medicine Analysis
54← Slide →
Obesity and metabolic syndrome are clear predictors of chronic kidney disease largely due to the potentiation of
chronic inflammation by insulin resistance. In addition, the lipoprotein abnormalities, increased hemodynamics,
hypercoagulability and vascular dysfunction associated with metabolic syndrome have all been implicated as causative
for renal disease. Biomarkers for renal dysfunction (i.e., IL6, TNF-α, NGAL, KIM-1, VEGF etc.) as well as significant
albuminuria , elevated free fatty acids with oxidative stress, and histological analysis have shown the ZDSD rat to exhibit
nephropathy that closely mimics that observed in obese insulin resistant patients.
Renal Injury
55
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Experimental details
• Male ZDSD rats were placed on a high-fat diet (RD12468) between 17 and 19 weeks of age. 15 out of 21 animals in this experiment developed diabetes during this period (this is usually 90%+).
• Rules Based Medicine panels (Rat Metabolic MAP, Rat Kidney MAP and RodentMAP™) were run on serum samples that were collected:
– before diabetes developed (14 weeks)
– while diabetic on the high fat diet (18 weeks) and
– one week after they were taken off the high fat diet (20 weeks).
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Neutrophil Gelatinous Associated Protein
•also called lipocalin2
•levels up-regulated during inflammation
•protective protein can trigger nephrogenesis
•associated with obesity, insulin resistance and hyperglycemia
57
Serum BioMarkers of Renal Disease
Age (weeks)
14 18 20
Seru
m N
GA
L (
ng/m
l)
200
400
600
800
1000
1200
1400 Sprague-Dawley (5)
ZDSD (6)
Diabetic ZDSD (15)
compared to SD
14 weeks = non-diabetic18 weeks = on diabetogenic diet one week20 weeks = off diabetogenic diet one week
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Beta-2-microglobulin
•a protein present in all nucleated cells which is normally reabsorbed in renal tubules
•increased circulating levels indicate glomerular membrane disease and inflammation
58
Age (weeks)
14 18 20
Se
rum
be
ta-2
-mic
roglo
bu
lin (
ug/m
l)
50
55
60
65
70
75
80
Sprague-Dawley (5)
ZDSD (6)
Diabetic ZDSD (15)
#
compared to SD
# Diabetic vs. non-diabetic
Serum BioMarkers of Renal Disease
14 weeks = non-diabetic18 weeks = on diabetogenic diet one week20 weeks = off diabetogenic diet one week
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Age (weeks)
14 18 20
Se
rum
Kim
-1 (
ng/m
l)
0.00
0.05
0.10
0.15
0.20
0.25
0.30
Sprague-Dawley (5)
ZDSD (6)
Diabetic ZDSD (15)
#
compared to SD
# diabetic vs. non-diabetic
Kidney-injury molecule-1
•a membrane protein, not normally present but appears in urine in response to acute kidney tubular injury
•highly sensitive predictor of renal injury when elevated in urine
59
Serum BioMarkers of Renal Disease
14 weeks = non-diabetic18 weeks = on diabetogenic diet one week20 weeks = off diabetogenic diet one week
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Age (weeks)
14 18 20S
eru
m G
ST
-alp
ha (
ng/m
l)
0
10
20
30
40
50
60
Sprague-Dawley (5)
ZDSD (6)
Diabetic ZDSD (15)
compared to SD
#
# Diabetic vs. non-diabetic
Glutathione-S-transferase-alpha
•enzyme that reduces toxin levels by conjugation with glutathione
•localized in proximal convoluted tubules, medullary tubules and loop of Henle
•in diabetes, hyperglycemia triggers oxidative stress which increases the renal excretion of this enzyme and therefore removes this protective function and increases blood levels.
•Biomarker for tubular kidney disease
60
Serum BioMarkers of Renal Disease
14 weeks = non-diabetic18 weeks = on diabetogenic diet one week20 weeks = off diabetogenic diet one week
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Clusterin
•(apolipoprotein J) is a protein highly correlated with apoptosis and the clearance of cellular debri
•It is elevated in glomeruli and tubules of diabetic kidneys
61
Serum BioMarkers of Renal Disease
Age (weeks)
14 18 20
Se
rum
Clu
ste
rin
(
g/m
l)
100
200
300
400
500
Sprague-Dawley (5)
ZDSD (6)
Diabetic ZDSD (15)
#
# Diabetic vs. non-diabetic
comapred to SD
14 weeks = non-diabetic18 weeks = on diabetogenic diet one week20 weeks = off diabetogenic diet one week
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von Willebrand factor
•required for platelet adhesion, makes platelets "sticky“•vWF binds inactive Factor VIII, protecting it from degradation•defiency leads to bleeding disorders•increased levels predispose to stroke•increases precede microalbuminuria in diabetic nephropathy
62
Age (weeks)
14 18 20
Se
rum
vo
n W
ille
bra
nd
Fa
cto
r (n
g/m
l)
0
50
100
150
200
250
300
Sprague-Dawley (5)
ZDSD (6)
Diabetic ZDSD (15)
compared to SD
#
#
# Diabetic vs. non-diabetic
Serum BioMarkers of Renal Disease
14 weeks = non-diabetic18 weeks = on diabetogenic diet one week20 weeks = off diabetogenic diet one week
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Experiment 4Diabetic Nephropathy,
EM of Glomerular Pathology
63← Slide →
Experimental details
• Male ZDSD rats allowed to become spontaneously diabetic.
• Animals were terminated by perfusion at about 35 weeks of age. We evaluated the following groups:
– Control CD rats
– ZDSD rats that had been diabetic from 12-13 weeks
– ZDSD rats that had been diabetic from 16-17 weeks
• Took pictures of glomerular capillaries and BM– Measured GBM thickness
– Evaluated podocyte morphology
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65
Glomerular CapillaryControl, Age Matched Diabetic, 12 Weeks
65
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Glomerular Capillary, Basement MembraneControl, Age Matched Diabetic, 12 Weeks
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Glomerular Capillary, Basement MembraneControl, Age Matched Diabetic, 12 Weeks
67
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68
Glomerular CapillaryControl, Age Matched Diabetic, 16.5 Weeks
68
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Glomerular Capillary, Basement MembraneControl, Age Matched Diabetic, 16.5 Weeks
• Male ZDSD rats synchronized to become diabetic by feeding them Purina 5SCA.
• Animals were put on 5SCA at 19 weeks of age and were diabetic by 20 weeks of age. They were monitored until they were 47 weeks old. We evaluated the following groups:
– ZDSD rats that had been diabetic for 27 weeks (14)
– ZDSD rats that failed to become diabetic (4)
• Graphed terminal data and evaluated pictures of glomeruli and other kidney pathology
Reference: Skeletal changes associated with theonset of type 2 diabetes in the ZDF and ZDSDrodent models. Susan Reinwald, Richard G.Peterson, Matt R. Allen, and David B. Burr. Am JPhysiol Endocrinol Metab 296: E765–E774, 2009.
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81← Slide →
0
100
200
300
400
500
600
700
800
7 9 11 13 15 17 19 21 23 25 27 29 31 33
Glu
cose
(m
g/d
l)
Age (weeks old)
ZDSD (diabetic)
Control (non-diabetic)
*
*
*
***
Reference range for controls
A
0
100
200
300
400
500
600
700
800
23 25 27 29 31 33
Age (weeks old)
ZDF fa/fa (diabetic)
ZDF fa/+ (non-diabetic)
B
Comparative Glucose Concentrations
Despite a later increase in blood glucose levels in the ZDSD rats, by 21-wks-old the average glucose concentrations are >500 mg/dl.
Mean ±SEM
Mean ±SEMn=12-17/group
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82← Slide →
L4 Vertebra – dimensions not affected by
differences in growth in ZDSD rats
0
1
2
3
4
5
6
7
8
ZDF (fa/fa) ZDF (+/fa)
L4
ve
rte
bra
l b
od
y h
eig
ht (m
m)
p<0.05
0
1
2
3
4
5
6
7
8
ZDSD Controls
L4
ve
rte
bra
l b
od
y h
eig
ht (m
m)
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
ZDF (fa/fa) ZDF (+/fa)
L4
cro
ss-s
ect
ion
al a
rea
(m
m)
p<0.05
0.27
0.28
0.29
0.30
0.31
0.32
0.33
0.34
0.35
ZDSD Controls
L4
cro
ss-s
ect
ion
al a
rea
(m
m)
Mean ±SEMn=12-17/group
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83← Slide →
Structural Properties L4 vertebrae
0
10
20
30
40
50
60
70
80
90
ZDF (fa/fa) ZDF (+/fa)
En
erg
y to
Ultim
ate
Lo
ad
(m
J)
*
ZDF ZDSD
p < 0.001 p < 0.005
p < 0.050 p < 0.001
p < 0.001 p < 0.001
p <0.050 p < 0.010
0
10
20
30
40
50
60
70
80
90
ZDSD Controls
En
erg
y to
Ultim
ate
Lo
ad
(m
J)
*
P-values for differences in diabetic rats vs. respective controls
[Biomechanical Test – axial compression]
Yield Force, N
Stiffness, N/mm
Ultimate Load, N
Energy to Ultimate Load, mJ
Mean ±SEMn=12-17/group
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84← Slide →
Material Properties L4 vertebrae
Ultimate Stress, N/mm2
Modulus, N/mm2
Toughness, mJ/mm3
Postyield Toughness, mJ/mm3
ZDF ZDSD[Parameters Normalized to BV/TV]
p < 0.051 p < 0.050
p < 0.050 p < 0.050
p = 0.657 p < 0.051
p = 0.224 p < 0.101
P-values for differences in diabetic rats vs. respective controls
0.0
0.5
1.0
1.5
2.0
2.5
ZDF (fa/fa) ZDF (+/fa)
To
ug
hn
ess/[B
V/T
V] (m
J/m
m3
)
0.0
0.5
1.0
1.5
2.0
2.5
ZDSD Controls
Tou
gh
ne
ss/[B
V/T
V] (
mJ/m
m3
)
*Mean ±SEMn=12-17/group
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85← Slide →
Glycated hemoglobin
0
1
2
3
4
5
6
7
8
9
10
ZDF (fa/fa) ZDF (+/fa)
Hb
A1
c (
%)
*
0
1
2
3
4
5
6
7
8
9
10
ZDSD Controls
Hb
A1
c (
%)
*
Evidence of a high level of non-enzymatic glycation (NEG, or cross-linking) occurring in the ZDSD rats.
The large decline in vertebral mechanical toughness in the ZDSD model may be attributable to an accumulation of NEGs in the bone collagen matrix. This possibility is currently under investigation. Mean ±SEM
n=12-17/group Return to Index
86← Slide →
Defective Wound Healing in ZDSD
• Wound was made with 6mm punch
• Animals were followed and pictures were taken on days 1, 4, 7, 9, 11 and 14
• Wounds were analyzed by evaluating and measuring the healing process. The diameter of open wound or thin skin (reddish in color) was measured
• Data were graphed
• There is a statistical difference in wound healing between ZDSD (diabetic and non-diabetic) and SD rats. Return to Index
87← Slide →
88
Control SD animals233019
Day 11
Day 14
233023
Day 11
Day 14
Diabetic ZDSD animals233003
233006Day 11
Day 14
Day 11
Day 14
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88← Slide →
Scale in mm is to the left of each picture. There is a visible difference in healing at 14 days.
Wound Healing in the ZDSD RatArea of Incompletely Healed Wound
Day of Study
Inco
mp
lete
ly H
eale
d
Wo
un
d A
rea, m
m
1 4 7 911
14 1 4 7 9
11
14 1 4 7 9
11
14
0
2
4
6
8 Diabetic ZDSD
Non-Diabetic ZDSD
Non-Diabetic SD*
*
*
*
*
This figure demonstrates the wound healing in the three groups of animals. There were no differences between diabetic and non-diabetic ZDSD animals. There were several statistically significant differences between the SD group and the ZDSD groups, which were particularly dramatic at day 14 (*). Return to Index
89← Slide →
The dysregulation of hemostasis is a common feature of metabolic syndrome and T2DM. Endothelial dysfunction, platelet hyperactivity, high platelet
count,hypercoagulability and decreased fibrinolysis have all been positively correlated with insulin resistance. Elevated levels of markers of endothelial
(PAI-1)have been reported in patients with this syndrome. Similarly, many markers known to indicate a prothrombotic state, including PAI-1, sVCAM, VWF, have been observed in ZDSD rats. Interestingly, an elevation in thrombopoeitin
was also noted in ZDSD and may indicate an over-production of platelets.
Pro-thrombotic environment.
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90← Slide →
Vascular endothelial growth factor
•promotes angiogenesis
•increased in atherosclerosis
•increased in diabetic retinopathy
•contributes heavily to renal endothelial dysfunction
•elevated with insulin resistance
Age (weeks)
14 18 20
Se
rum
VE
GF
(p
g/m
l)
150
200
250
300
350
400
450
Sprague-Dawley (5)
ZDSD (6)
Diabetic ZDSD (15)
compared to SD
Serum BioMarkers of Coagulation and Vascular Disease
Confidential
14 weeks = non-diabetic18 weeks = on diabetogenic diet one week20 weeks = off diabetogenic diet one week
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91← Slide →
von Willebrand factor
•required for platelet adhesion
•binds inactive Factor VIII, protecting it from degradation
